Volume 15 Number 10 October 2013 pp. 1196–1206 1196 www.neoplasia.com

Sym004, a Novel EGFR Mari Iida*, Toni M. Brand*, Megan M. Starr*, Chunrong Li*, Evan J. Huppert*, Neha Luthar*, † † † Mixture, Can Overcome Acquired Mikkel W. Pedersen , Ivan D. Horak , Michael Kragh Resistance to Cetuximab1 and Deric L. Wheeler* *Department of Human Oncology, University of Wisconsin School of Medicine and Public Health, Wisconsin Institutes for Medical Research, Madison, WI; †Symphogen A/S, Lyngby, Denmark

Abstract The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in a variety of human cancers. is an anti-EGFR that has been approved for head and neck and colorectal cancer treatment, but many patients treated with cetuximab don’t respond or eventually acquire resistance. To determine how tumor cells acquire resistance to cetuximab, we previously developed a model of acquired resis- tance using the non–small cell lung cancer line NCI-H226. These cetuximab-resistant (CtxR) cells exhibit increased steady-state EGFR expression secondary to alterations in EGFR trafficking and degradation and, further, retained dependence on EGFR signaling for enhanced growth potential. Here, we examined Sym004, a novel mixture of directed against distinct on the extracellular domain of EGFR, as an alternative therapy for CtxR tumor cells. Sym004 treatment of CtxR clones resulted in rapid EGFR degradation, followed by robust inhibition of cell proliferation and down-regulation of several mitogen-activated protein kinase pathways. To determine whether Sym004 could have therapeutic benefit in vivo, we established de novo CtxR NCI-H226 mouse xenografts and subsequently treated CtxR tumors with Sym004. Sym004 treatment of mice harboring CtxR tumors resulted in growth delay compared to mice continued on cetuximab. Levels of total and phospho-EGFR were robustly de- creased in CtxR tumors treated with Sym004. Immunohistochemical analysis of these Sym004-treated xenograft tumors further demonstrated decreased expression of Ki67, and phospho-rpS6, as well as a modest increase in cleaved caspase-3. These results indicate that Sym004 may be an effective targeted therapy for CtxR tumors.

Neoplasia (2013) 15, 1196–1206

Abbreviations: CtxR, cetuximab-resistant; CtxS, cetuximab-sensitive; EGFR, epidermal growth factor receptor; ERK1/2, extracellular signal–regulated kinases 1 and 2; HP, parental NCI-H226 cells; HNSCC, head and neck squamous cell carcinoma; i.p., intraperitoneal; MAPK, mitogen-activated protein kinase; mAb, monoclonal antibody; mCRC, metastatic colorectal cancer; NSCLC, non–small cell lung cancer; siRNA, small interfering RNA Address all correspondence to: Deric L. Wheeler, PhD, Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, 1111 Highland Avenue, WIMR 3159, Madison, WI 53705. E-mail: [email protected] 1The project described was supported, in part, by grant UL1TR000427 from the Clinical and Translational Science Award program, through the National Institutes of Health (NIH) National Center for Advancing Translational Sciences, grant RSG-10-193-01-TBG from the American Cancer Society (D.L.W.), grant 888157 from Symphogen A/S (D.L.W.), and NIH grant T32 GM08.1061-01A2 from Graduate Training in Cellular and Molecular Pathogenesis of Human Diseases (T.M.B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Conflict of interest: D.L.W. holds a laboratory research agreement with Symphogen A/S. M.W.P., I.D.H., and M.K. are employed by Symphogen A/S. No potential conflicts of interest were disclosed by other authors. Received 5 September 2013; Revised 25 September 2013; Accepted 25 September 2013 Copyright © 2013 Neoplasia Press, Inc. All rights reserved 1522-8002/13/$25.00 DOI 10.1593/neo.131584 Neoplasia Vol. 15, No. 10, 2013 Sym004 Can Overcome Resistance to Cetuximab Iida et al. 1197

Introduction In this study, we hypothesized that cells with acquired resistance The epidermal growth factor receptor (EGFR) is a member of the to cetuximab retain growth dependency on the EGFR and would human epidermal growth factor receptor (HER) family of receptor therefore benefit from therapeutic agents that promote EGFR degra- tyrosine kinases that consists of four members: EGFR (ErbB1/HER1), dation. We found that Sym004 treatment delays the emergence of HER2/neu (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Stimulation acquired resistance to cetuximab in NCI-H226 cells both in vitro of EGFR through ligand binding promotes receptor homodimerization and in vivo. Down-regulation of several MAPK pathways was also or heterodimerization with other HER family receptors, leading to acti- observed in response to Sym004 treatment. The results presented vation of its intrinsic tyrosine kinase [1,2]. Activation of EGFR initiates herein suggest that tumors with acquired resistance to cetuximab various signaling cascades, one of which is the mitogen-activated protein can thus be successfully targeted with the novel anti-EGFR thera- kinase (MAPK) network. MAPKs belong to a group of intracellular ser- peutic Sym004. ine/threonine protein kinases that consist of various members, including the extracellular signal–regulated kinases 1 and 2 (ERK1/2) and p38 MAPKs (alpha, beta, gamma, delta). Upon activation of EGFR, the Materials and Methods MAPK pathway is initiated through the promotion of Ras binding to guanosine triphosphate (GTP), which in turn activates RAF kinases Cell Lines MAPK/extracellular signal-regulated kinases (MEK), and MAPKs. The human NSCLC cell line NCI-H226 was provided by Drs J. Minna MAPKs can subsequently activate numerous transcription factors such and A. Gazdar (University of Texas Southwestern Medical School, as c-Fos, c-Myc, and Elk-1 and numerous protein kinases such as Dallas, TX). The cells were maintained in 10% FBS in RPMI-1640 ribosomal S6 kinase 1 (RSK1) [3]. Collectively, the stimulation of the (Mediatech, Inc, Manassas, VA) with 1% penicillin and streptomycin. MAPK pathway by EGFR has been shown to greatly enhance cell The development of CtxR cells has been described previously [10]. proliferation, angiogenesis, invasion, and metastasis in cancer cells. Aberrant expression or activity of EGFR has been identified in Small Interfering RNA and Transfection many human epithelial cancers, including non–small cell lung cancer For small interfering RNAs (siRNAs), CtxR cells (HC1, HC4, and (NSCLC). Therefore, targeting EGFR has been intensely pursued HC8) were transiently transfected with siEGFR (ON-TARGETplus, over the last three decades as a cancer treatment strategy. One SMART pool #L-003114-00; Dharmacon, Lafayette, CO) using approach to inhibit the activation of EGFR is through the use of Lipofectamine RNAiMAX according to the manufacturer’s instruc- monoclonal antibodies (mAbs) that bind the extracellular domain tions (Invitrogen, Carlsbad, CA). The nontargeting siRNA (ON- of EGFR to block natural ligand binding. Cetuximab (IMC-225, TARGETplus Non-targeting Pool, #D-001810-10) was obtained Erbitux) is a human/murine chimeric mAb that was developed to from Dharmacon as a control. Cells were then lysed for analysis of bind to the extracellular domain III of EGFR. This interaction par- protein knockdown by immunoblot analysis after siRNA transfection. tially blocks the ligand-binding domain and sterically hinders the correct extended conformation of the dimerization arm on domain II [4]. The Food and Drug Administration has approved cetuximab Materials treatment for patients with metastatic colorectal cancer (mCRC) and Cetuximab (IMC-225, Erbitux) was purchased from the Univer- head and neck squamous cell carcinoma (HNSCC). However, while sity of Wisconsin Pharmacy. Sym004 was generously provided by cetuximab has shown beneficial antitumor effects in these cancers, Symphogen A/S (Lyngby, Denmark). the majority of patients who initially respond eventually acquire resistance [1,5,6]. Antibodies Numerous efforts have been undertaken to define the molecular All antibodies were purchased from commercial sources as indicated mechanisms of acquired resistance to cetuximab [7–14]. Our laboratory below: EGFR, pEGFR (Y1173), and HRP-conjugated goat anti-rabbit has established panels of cetuximab-resistant (CtxR) clones from the IgG and goat anti-mouse IgG were obtained from Santa Cruz Bio- NSCLC cell line NCI-H226 by exposing these cells in vitro to increas- technology, Inc (Dallas, TX). pEGFR (Y1045), pEGFR (Y1068), ing concentrations of cetuximab. Using this model, we have shown pERK1/2 (T202/Y204), p44/42 ERK1/2, p-rpS6 (S235/236), rpS6, that impaired EGFR internalization and degradation lead to increased p-c-RAF (S289/296/301), c-RAF, pRSK1 (S380), RSK1, p-p38 EGFR surface level expression, increased EGFR kinase activity, and (T180/Y182), and p38 were obtained from Cell Signaling Technology dependence on EGFR induced signaling pathways [7]. These data (Danvers, MA). pERK1/2 (T202/Y204/T185/Y187) was purchased suggest that EGFR remains a molecular target in this setting and that from Abcam (Cambridge, MA). α-Tubulin was purchased from new therapeutics that can initiate the rapid internalization and robust Calbiochem (Billerica, MA). degradation of EGFR may be a potent anticancer strategy. Sym004 is a novel 1:1 mixture of a pair of mAbs (992 and 1024) directed against nonoverlapping epitopes on EGFR [15,16]. Sym004 Cell Proliferation Assay inhibits cancer cell growth and survival by blocking ligand binding, This was performed as previously described [10]. Cells were seeded receptor activation, and downstream signaling. Unlike cetuximab, in six-well plates. Following treatment, monolayers were analyzed by Sym004 induces rapid and efficient removal of the receptor from crystal violet assay. All treatments were performed in triplicate. Cells the cell surface by triggering EGFR internalization and degradation. were seeded at 2000 cells per well in 100 μl of media on a 96-well Additionally, Sym004 has been shown to elicit antibody-dependent plate, grown for 24 hours, and then treated with drug for 72 hours cellular cytotoxicity and complement-dependent cytotoxicity in vivo before analysis using the Cell Counting Kit 8 (Dojindo Molecular [16], while cetuximab has been shown to elicit antibody-dependent Technologies, Rockville, MD) according to the manufacturer’s cellular cytotoxicity only [16–19]. instructions. All treatments were performed in quadruplicate. 1198 Sym004 Can Overcome Resistance to Cetuximab Iida et al. Neoplasia Vol. 15, No. 10, 2013

Cell Growth Assay In Vivo Sym004). The dose of cetuximab and Sym004 for the experiment Athymic nude mice (4- to 6-week-old males) were obtained from was 1, 5, 20, and 50 mg/kg and twice a week by i.p. injection. Tumor Harlan Laboratories (Indianapolis, IN). All animal procedures and volume measurements were evaluated by digital calipers and calcu- maintenance were conducted in accordance with the institutional lated by the formula (p)/6 × (large diameter) × (small diameter)2. guidelines of the University of Wisconsin. Mice were randomized into treatment or control groups. Parental NCI-H226 (HP), HC1, Mouse CtxR Human Tumor Xenografts or HC4 cells were injected in the dorsal flank of the mouse at respec- Mice were injected with NCI-H226 (2 × 106 cells), and tumors tive day 0 (1 × 106 cells). Once tumors reached 250 mm3, mice were were allowed to grow to 100 mm3. All mice were randomized to started on 0.5 mg of cetuximab treatments twice a week by intra- treatment or control groups and treated with 1 mg/mouse (40 mg/kg) peritoneal (i.p.) injection. Tumor volume measurements were evalu- of either cetuximab or IgG i.p. twice weekly. Tumors were monitored ated by digital calipers and calculated by the formula (p)/6 × (large for cetuximab resistance that was defined as marked tumor growth in diameter) × (small diameter)2. the presence of continued cetuximab therapy. Once CtxR tumors reached a volume of ∼1000 mm3, mice were grouped according to Immunoblot Analysis similar time points of resistance. At this point, each mouse was treated Whole-cell protein lysate was obtained using Tween-20 lysis buffer with either cetuximab (1 mg) or Sym004 (20 mg/kg) i.p. twice weekly. (50 mM Hepes, pH 7.4, 150 mM NaCl, 0.1% Tween-20, 10% glyc- Tumor volume measurements were evaluated by digital calipers and 2 erol, 2.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM Na3VO4, calculated by the formula (p)/6 × (large diameter) × (small diameter) . 1 mM PMSF, 1 mM beta-glycerophosphate (BGP), and 10 μg/ml leupeptin and aprotinin). Immunoblot analysis was conducted as Mouse Tumor Collection and Protein Isolation previously described [20]. Tumors were collected 3 hours after the last cetuximab or Sym004 treatment. Mice were sedated using isoflurane mixed with oxygen Bromodeoxyuridine Cell Cycle Distribution Analysis until unconscious. Mice were killed by cervical dislocation, and tumors were promptly collected, washed in PBS, and frozen with isopentane on Cells were plated at a density of 800,000 per 100 mm2 plate and dry ice. Whole-cell protein lysates from tumor samples were obtained allowed to adhere overnight. The cells were treated with vehicle, withNP-40lysisbuffer(50mMHepes,pH7.4,150mMNaCl, 20 μg/ml cetuximab, or 20 μg/ml Sym004 for 24 hours. On the 1% NP-40, 0.5% deoxycholic acid, 10% glycerol, 2.5 mM EGTA, following day, the cells were pulsed with 10 μM bromodeoxyuridine 1mMEDTA,1mMDTT,1mMPMSF,and10μg/ml leupeptin for 1 hour. The cells were harvested by trypsinization, washed with and aprotinin), homogenized by 10 strokes in a tightly fitting Dounce cold phosphate-buffered saline (PBS), and fixed with 70% ethanol homogenizer, and quantified. Protein quantitation and immunoblot for 20 minutes. The cells were then labeled with a fluorescein analysis were performed as stated above. isothiocyanate–conjugated mouse anti-bromodeoxyuridine antibody and processed according to the manufacturer’s recommendations Immunohistochemistry (BD Pharmingen, San Jose, CA). The cells were analyzed by flow Tumor tissue samples were collected from xenograft tumors. Tumor cytometry (BD FACScan). ModFit Software (Verity Software House, samples were fixed in 4% formaldehyde/PBS paraffin-embedded and Topsham, ME) was used to analyze the data. section. Sections were heated in 10 mM citrate buffer (pH 6.0) for EGFR, Ki67, and phospho-rpS6 or in boric acid buffer (pH 8.0) for Phospho-MAPK Array cleaved caspase-3 by Decloaking Chamber. Samples were incubated Cell lines were analyzed in the panel of phosphorylation profiles of with rabbit anti-EGFR (Abcam; ab52894, 1:200), rabbit anti-Ki67 MAPK and other serine/threonine kinases after treatment with (Abcam; ab66155, 1:1000), rabbit cleaved caspase-3 (Biocare, Con- Sym004 (Human Phospho-MAPK, ARY002B; R&D Systems, cord, CA; CP229b, 1:100), and rabbit phospho-rpS6 (S235/236; Cell Minneapolis, MN). This array specifically screens for relative levels Signaling Technology; 1:400). Sections were stained by BrightVision of phosphorylation of 26 individual proteins including 9 MAPKs rabbit/HRP (Immunologic, Duiven, The Netherlands; and other intracellular proteins involved in cellular proliferation. DPVR110HRP) or LSAB kit/HRP (Dako, Carpinteria, CA). Anti- μ After treatment with or without Sym004 (50 g/ml), cell lysates were body binding was revealed by addition of 3,3′-diaminobenzidine sub- incubated with the membrane. Thereafter, a cocktail of biotinylated strate. Tissues were counterstained with Mayer’s hematoxylin (Thermo detection antibodies, streptavidin-HRP, and chemiluminescent Fisher Scientific, Waltham, MA) and were examined using an Olympus detection reagents was used to detect the phosphorylated protein. BX51 microscope. The immunohistochemical staining was judged as The relative expression of specific phosphorylated protein was deter- follows; Ki67: staining was analyzed with a PC-based image analysis mined following quantification of scanned images by ImageJ com- system (Leica Q500, Cambridge, United Kingdom). With this equip- pared with Sym004 and vehicle. ment the positively stained DAB reaction was measured and expressed as percentage of positive stained area in relation to total analyzed viable Mouse Xenograft Model tumor area. Cleaved Caspase-3: no positive cells, low numbers of Athymic nude mice (4- to 6-week-old males) were obtained from positive cells (1+) or moderate numbers of positive cells (2+). Harlan Laboratories. All animal procedures and maintenance were conducted in accordance with the institutional guidelines of the Uni- Results versity of Wisconsin. Mice were randomized into treatment or control groups. Mice were injected in the dorsal flank of the mouse CtxR Clones Have Increased EGFR Activity at respective day 0 (2 × 106 cells). Once tumors reached 200 mm3, We have previously reported that three CtxR clones (HC1, HC4, mice were started on their respective treatments (IgG, cetuximab, and HC8) display a robust CtxR phenotype when challenged with Neoplasia Vol. 15, No. 10, 2013 Sym004 Can Overcome Resistance to Cetuximab Iida et al. 1199 increasing doses of cetuximab as compared to HP parental control siRNAs targeting EGFR. All three CtxR clones displayed prolif- cells at 72 hours [10]. To examine the stability of the resistant pheno- eration inhibitory effects at 30 nM siEGFR (Figure 2A). Loss of type in vivo, we inoculated 1 × 106 cells of both HP and CtxR clones EGFR expression resulted in loss of activated c-RAF (S289/296/ HC1 and HC4 into the dorsal flank of athymic nude mice. Tumors 301) and ERK1/2 (T202/Y204) kinases, demonstrating that were allowed to grow to 250 mm3 and then treated with 0.5 mg of MAPK pathway activation is dependent on EGFR activity. Next, cetuximab/mouse i.p. twice weekly for the time indicated. As shown we treated CtxR clones with 20 μg/ml cetuximab or increasing doses in Figure 1A,CtxR clones established in culture maintained their of Sym004 for 72 hours. All CtxR clones demonstrated statistically resistant phenotype in the xenograft model system. CtxR clones dis- significant, dose-dependent inhibition of proliferation in response played increased steady-state expression of EGFR and EGFR phos- to Sym004 treatment compared to cetuximab or vehicle treatment phorylation (Y1173, Y1068, and Y1045). Increased phosphorylation (Figure 2B). levels of downstream signaling molecules, including ERK1/2 (T202/ Y204/T185/Y187), p38 (T180/Y182), RSK1 (S380), c-RAF (S289/ 296/301), and rpS6 (S235/236), were detected by immunoblot analy- Sym004 Effectively Degrades EGFR in CtxR Clones, sis in the CtxR clones (HC1, HC4, HC8) relative to HP control cells whereas Cetuximab Has No Effect on Total and (Figure 1B). Taken together, these results confirm the establishment Phosphorylated EGFR Levels of stable CtxR clones that exhibit robust activation of the EGFR To investigate if inhibition of cell proliferation by Sym004 is due signaling cascade. to EGFR degradation, we examined the expression levels of EGFR in CtxR clones treated with 20 μg/ml cetuximab or Sym004 (0.1, 1, 20, 50 μg/ml) for 24 hours (Figure 3A). Since our previous study showed CtxR Clones Are Dependent on EGFR for that small molecule tyrosine kinase inhibitors (TKIs) decreased EGFR Proliferative Potential phosphorylation at tyrosine 1173 (Y1173), we chose this site for evalua- To determine if CtxR clones remained dependent on EGFR for tion of Sym004 [10]. Both activated and total levels of EGFR were enhanced growth potential, we performed proliferation assays using decreased at all doses of Sym004 examined in CtxR clones tested;

Figure 1. CtxR clones have increased EGFR activity. (A) CtxR clones are resistant to cetuximab in vivo. Male athymic nude mice were injected subcutaneously with 1 × 106 CtxS HP or CtxR cells (HC1 or HC4) into the dorsal flank. Once tumors reached a volume of 250 mm3, mice were treated with 0.5 mg of cetuximab twice weekly. Tumor diameters were measured serially with calipers. Tumor volumes were calculated and graphed as fold change in tumor volume + S.E. for each cell line. (B) CtxR cells have increased EGFR activity and phos- phorylation levels of downstream signaling molecules. HP and CtxR clones (HC1, HC4, HC8) were harvested, and protein lysates were fractionated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblot analysis for the indi- cated proteins. α-Tubulin was examined as a loading control for each immunoblot analysis. 1200 Sym004 Can Overcome Resistance to Cetuximab Iida et al. Neoplasia Vol. 15, No. 10, 2013

Figure 2. CtxR clones are dependent on EGFR for proliferative potential. (A) siEGFR inhibits the proliferation of CtxR clones. CtxR clones were plated and treated with 30 nM of EGFR siRNA or 30 nM nontargeting (NT) siRNA. Proliferation was measured at 72 hours after treatment using the cell counting kit-8 (CCK-8) proliferation assay described in the experimental procedures. Data points are represented as means ± SEM (n = 4). *P ≤ .05. CtxR clones (HC1, HC4, HC8) were harvested, and protein lysates were fractionated on SDS-PAGE, followed by immunoblot analysis for the indicated proteins. α-Tubulin was used as a loading control. (B) Sym004 inhibits the proliferation of CtxR clones. HP and CtxR cell lines (HC1, HC4, HC8) were plated and allowed to adhere for 24 hours before vehicle, cetuximab (20 μg/ ml), or Sym004 treatment: 0.1, 1, 20, or 50 μg/ml. Proliferation was measured at 72 hours after drug treatment using the crystal violet assay and plotted as a percentage of growth relative to untreated control cells. Data points are represented as means ± SEM (n = 3). increasing dosing of Sym004 led to more potent decreases in EGFR The total and phosphorylated EGFR levels were inhibited by Sym004 levels. In contrast, cetuximab had no impact on total EGFR levels. at all three time points, whereas cetuximab had no effect on total EGFR In a time course experiment, CtxR clones were treated with a single dose and slightly activated EGFR in HC1 and HC4 clones at 24 hours of 20 μg/ml Sym004 or cetuximab for 24, 48, or 72 hours (Figure 3B). post treatment. These results demonstrate that Sym004 can effectively

Figure 3. Sym004 effectively degrades EGFR in CtxR clones, whereas cetuximab has no effect on total and phosphorylated EGFR levels. (A) Sym004 downregulates total and phosphorylated EGFR in CtxR clones. CtxR clones (HC1, HC4, HC8) were treated with vehicle, cetuximab (20 μg/ml), or Sym004 (0.1, 1, 20, or 50 μg/ml) for 24 hours. Whole-cell protein lysates were fractionated on SDS-PAGE followed by immunoblot analysis for the indicated proteins. α-Tubulin was used as a loading control. (B) Total and phosphorylated forms of EGFR remain downregulated 72 hours post Sym004 treatment in CtxR clones. Cells were treated with a single dose of 20 μg/ml cetuximab or 20 μg/ml Sym004 for 24, 48, or 72 hours. Cell lysates were prepared, and EGFR and phospho-EGFR were determined by immunoblot analysis. Neoplasia Vol. 15, No. 10, 2013 Sym004 Can Overcome Resistance to Cetuximab Iida et al. 1201

Figure 4. Multiple MAPK effector molecules are inhibited in CtxR clones by Sym004 treatment. (A) Sym004 inhibited multiple downstream MAPK effector molecules detected through phospho-kinase array. After treatment with Sym004 (50 μg/ml), cells were collected and cell extracts were incubated with membranes containing antibodies to 26 individual proteins. The membranes were washed and incubated with a cocktail of biotinylated detection antibodies, streptavidin-HRP, and chemiluminescent detection reagents to measure the levels of phosphorylated protein. Quantitation of phosphorylated proteins was completed using scanned images from ImageJ software. Data points are represented as the mean of duplicate spots. Cell extracts were also fractionated on SDS-PAGE, followed by immunoblot analysis for the indicated proteins. α-Tubulin was examined as a loading control for each immunoblot analysis. (B) Sym004 can induce a G1-phase cell cycle arrest in CtxR clones. Cells were treated with 20 μg/ml Sym004 for 24 hours, and cell cycle phase distribution was analyzed as described in the Materials and Methods section. Data points are represented as means ± SEM (n =3).*P <.05. downregulate EGFR in all CtxR clones for prolonged periods of time treatment on MAPK signaling in each clone using a Human Phospho- post treatment. MAPK Array. CtxR clones were treated with vehicle or Sym004 (50 μg/ml) for 24 hours. This Human Phospho-MAPK array includes Multiple MAPK Pathway Proteins Are Inhibited in 26 kinases, 9 of these are MAPKs. As illustrated in Figure 4A, Sym004 CtxR Cells by Sym004 Treatment decreased phosphorylation in three CtxR clones by averages of 80% CtxR cells exhibited an increase in EGFR expression and activa- for ERK1, 70% for p38γ, 40% for ERK2, 40% for RSK1, and 30% tion, as well as activated MAPK pathway proteins (Figure 1B). To for P38β, compared with the vehicle control. Consistent with the investigate potential mechanisms by which Sym004 elicits anti- array results, Western blot analysis indicated that phosphorylation proliferative effects in CtxR clones, we analyzed the effect of Sym004 of c-RAF (S289/296/301), ERK1/2 (T202/Y204/T185/Y187), 1202 Sym004 Can Overcome Resistance to Cetuximab Iida et al. Neoplasia Vol. 15, No. 10, 2013 p38 (T180/Y182), and RSK1 (Ser380) were robustly inhibited after xenografts at doses of 5 mg/kg or higher compared to the IgG control Sym004 treatment in all CtxR cells. group (Figure 5). Next, cell cycle phase distribution of CtxR clones was analyzed post Sym004 treatment. Flow cytometric analysis demonstrated that Sym004 Treatment of CtxR Tumors Leads to R In Vivo Sym004 induced a strong G1 arrest in Ctx cell lines (Figure 4B). Tumor Growth Delay Taken together, these data demonstrate that Sym004 can inhibit Next, we performed a series of mouse xenograft studies of a pre- EGFR signaling through the MAPK pathway, ultimately impacting viously developed model of de novo acquired resistance to cetuximab cell cycle progression in CtxR clones. [7]. To develop acquired resistance to cetuximab in vivo, we inocu- lated 40 mice with the NSCLC NCI-H226 cell line unilaterally 6 Both Cetuximab and Sym004 Delay Tumor Growth in with 2 × 10 tumor cells in the dorsal flank. Tumors were allowed Cetuximab-Sensitive H226 Cells to grow to 100 mm3, at which time 30 mice were treated with To investigate the effects of Sym004 in vivo, mice bearing estab- cetuximab (1 mg/mouse) and 10 mice were treated with IgG control lished NCI-H226 [cetuximab-sensitive (CtxS) parental cell line] xeno- (1 mg/mouse) twice weekly by i.p. injection. IgG-treated tumors grafts were treated with vehicle control, cetuximab, or Sym004. Mice grew rapidly uninhibited, whereas cetuximab-treated animals dem- were injected in the dorsal flank on day 0 (2 × 106 NCI-H226 cells), onstrated tumor control and delayed growth. Tumors were moni- and once tumors reached an average volume of 200 mm3 (∼18 days), tored for the development of cetuximab resistance, defined as marked mice were randomly grouped. Cetuximab or Sym004 was admin- tumor growth in the presence of continued cetuximab therapy. Once R 3 istered through i.p. injection at a dose of 1, 5, 20, or 50 mg/kg Ctx tumors reached a volume of ∼1000 mm , mice were grouped R twice weekly for four consecutive weeks. Mouse weight was mea- according to similar size at the time of resistance detection. Ctx was sured weekly, and no discernible toxicity was observed in either the observed in 21 of 30 tumor xenografts (70%) treated with cetuximab, cetuximab or Sym004 treatment group. Treatment with either similar to previous studies from our laboratory [7]. Thus, a total of R cetuximab or Sym004 showed clear dose-dependent antitumor activ- nine Ctx mouse xenograft groups was selected for further study R ity, and both agents significantly delay tumor growth of CtxS H226 (21 mice in total). Upon establishment of Ctx mouse groups, one mouse was maintained on cetuximab (1 mg) therapy, while the other mouse (or mice) in the group was removed from cetuximab and started on 20 mg/kg Sym004 (i.p. twice weekly). The average tumor volume of mice treated with IgG alone is included in all groups for comparison purposes. Eight of 12 (67%) CtxR tumors treated with Sym004 demonstrated a tumor response compared to the cetuximab-treated mouse in each group, while four (33%) tumors failed to respond. In Figure 6A, we illustrate six of the nine groups where Sym004 delayed tumor growth more than 30 days. The black arrow designates the starting time point of Sym004 treatment. To further investigate the ability of Sym004 to effectively target EGFR in large tumors in vivo, we examined total and phospho-EGFR levels in individual tumors by immunoblot analysis (Figure 6B). Sym004-treated CtxR tumors had essentially undetectable levels of EGFR, whereas cetuximab-treated CtxR tumors retained significant levels of EGFR. These findings were very similar to the results presented in Figure 3A. Next, we verified these findings in tissue sections of the tumors by immunohistochemical analysis of EGFR, as well as markers for cell proliferation (Ki67) and apoptosis (cleaved caspase-3; Figure 6C ). Strong membrane and intracellular EGFR staining were observed in IgG- and cetuximab-treated CtxR tumors. Sym004-treated CtxR tumors showed moderate intracellular EGFR staining, and no or very limited membrane staining was seen in the majority of tumors examined. The highest numbers of Ki67-positive cells (18%) are seen in IgG followed by cetuximab-treated group (7%), and the lowest numbers are seen in Sym004-treated group (2%). Cleaved caspase-3 staining (black arrows) revealed that the most extensive expression was seen in samples treated with Sym004 followed by cetuximab-treated samples, and the most restricted expression was seen in IgG-treated samples. As a survival marker, phospho-rpS6 (S235/S236) was also stained in these CtxR tumors. CtxR tumors treated with Sym004 demonstrated promi- Figure 5. Both cetuximab and Sym004 can effectively control the nent inhibition of p-rpS6 compared to IgG or cetuximab-treated CtxR growth of CtxS mouse xenograft tumors. NCI-H226 cells were 3 tumors (Figure 6C ). These xenograft studies suggest that Sym004 can injected into mice, and tumors were allowed to grow to 200 mm . R All mice were randomized to treatment or control groups and treated effectively inhibit the growth of large Ctx tumors, providing strong with cetuximab (1, 5, 20, or 50 mg/kg), Sym004 (1, 5, 20, or 50 mg/kg), evidence for the use of Sym004 in the setting of acquired resistance or IgG (50 mg/kg) i.p. twice weekly. to cetuximab. Neoplasia Vol. 15, No. 10, 2013 Sym004 Can Overcome Resistance to Cetuximab Iida et al. 1203

Figure 6. Sym004 treatment delays the growth of CtxR xenograft tumors. (A) Growth-inhibitory effects of Sym004 in CtxR tumors in vivo. Mice were injected with NCI-H226, and tumors were allowed grow to 100 mm3. All mice were randomized to treatment or control groups and treated with either 1 mg/mouse (40 mg/kg) of cetuximab or IgG i.p. twice weekly. Tumors were monitored for cetuximab resistance, defined as marked tumor growth in the presence of continued cetuximab therapy. Once CtxR tumors reached a volume > 1000 mm3, mice were grouped according to similar time points of resistance. At this point, each mouse was treated with either cetuximab or Sym004 i.p. twice weekly. The black arrow designates the starting time point of Sym004 treatment. The average tumor volume of mice treated with IgG is included in all groups for comparison purposes. (B) Sym004-induced EGFR degradation in vivo. Immunoblot analysis of total and activated EGFR in CtxR xenograft tumors after cetuximab or Sym004 treatment. C, cetuximab; S, Sym004. (C) The degradation of EGFR in CtxR tumors corresponds with loss of proliferation and enhancement of apoptosis. CtxR tumor samples after cetuximab or Sym004 treatment in vivo were prepared and analyzed for EGFR, proliferation (Ki67), apoptosis (cleaved caspase-3), and phospho-rpS6 immuno- histochemistry. Black arrows denote cells positive for cleaved caspase-3. The percentage of Ki67-positive cells and the number of positive cells for cleaved caspase-3 expression are shown. 1204 Sym004 Can Overcome Resistance to Cetuximab Iida et al. Neoplasia Vol. 15, No. 10, 2013

Discussion ing that EGF could enhance resistance to cetuximab in numerous cancer EGFR is one of the most highly targeted receptors in oncology due cell lines; interestingly, researchers further showed that Sym004 treat- to its frequent overexpression and aberrant activation in numerous ment of the same cell lines yielded less ligand-induced resistance [15]. cancers. The anti-EGFR mAb therapy cetuximab is a Food and Drug Therefore, it seems plausible that Sym004-directed degradation Administration–approved treatment for mCRC [21] and HNSCC of EGFR in the current CtxR model might overcome compensatory [22] and has shown efficacy in other tumor types such as NSCLC up-regulation of ligand. [23,24]. While cetuximab has demonstrated clinical success, both Another mechanism by which Sym004-directed degradation of intrinsic and acquired resistance is commonly observed. To charac- EGFR may overcome CtxR is through the inhibition of EGFR terize the mechanisms of resistance to cetuximab in NSCLC, the cell nuclear translocation. Numerous studies have demonstrated that line NCI-H226 was treated with increasing doses of cetuximab until nuclear EGFR can enhance resistance to cetuximab [8], gefitinib resistant cell clones developed. In this model, we found that CtxR [31], radiation [32,33], and chemotherapy [34,35]. Previous research clones had increased EGFR expression and dependency on EGFR with the current model demonstrated that inhibition of nuclear for enhanced proliferative potential [10,11]. Therefore, we hypothe- EGFR could resensitize resistant clones to cetuximab [8]. Sym004- sized that Sym004, a novel anti-EGFR mAb mixture that leads to directed degradation of EGFR appears to deplete both the cell rapid internalization and degradation of the EGFR [15], may over- membrane component of EGFR, as well as nuclear EGFR, leading come resistance to cetuximab. The present study demonstrates that to effective knockdown of the EGFR signaling network. Thus, Sym004 can elicit potent antiproliferative effects in CtxR clones, Sym004 may play a vital role in overcoming resistance to gefitinib, which corresponded to the degradation of total EGFR. The loss of radiation, and chemotherapy as well. EGFR inhibited the activity of multiple MAPK signaling proteins The MAPK signaling pathway is one of the main pathways ema- R corresponding to G1 phase cell cycle arrest. De novo xenograft models nating from activated EGFR, and thus, Ctx clones express activated of NSCLC (NCI-H226) acquired CtxR further indicated that forms of MAPK pathway proteins (Figure 1B). Sym004 treatment Sym004 could significantly delay the growth of large CtxR tumors. inhibited two of the four major groups of conventional MAPKs, Collectively, these data suggest that Sym004 may be an invaluable including ERK1/2 and p38 MAPK, in addition to RSK1 and c-RAF treatment for EGFR-overexpressing CtxR tumors. (Figure 4A). RSK1 has specifically been shown to play an important Cetuximab is a human/murine chimeric mAb that binds to the role in promoting cell cycle progression past the G1 checkpoint through extracellular ligand-binding domain III of EGFR [4,25]. Cetuximab the phosphorylation of p27Kip1 and serum response factor, ultimately prevents EGFR ligand binding and sterically hinders dimerization enhancing cyclin D expression [36]. Therefore, inhibition of RSK1 with other HER receptors; thus, cetuximab has been shown to mayplayaroleintheG1 phase cell cycle arrest observed upon inhibit signaling cascades emanating from activated EGFR [4,26]. Sym004 treatment (Figure 4B). While the status of MAPK signaling Sym004 is a mixture of two EGFR-directed antibodies that bind to proteins were analyzed in this study, the functions of other proteins two distinct epitopes on domain III [15,16]. Sym004 can inhibit were likely modulated as well, and therefore, the antiproliferative effects EGFR activation and downstream signaling pathways but in contrast of Sym004 observed here may not be solely due to MAPK inhibition. to cetuximab, it can induce robust EGFR cross-links on the cell surface Additionally, while Sym004 yielded potent antiproliferative effects leading to effective internalization and degradation of the receptor in vitro, apoptosis was only minimally increased (data not shown). [15,16]. In the present study, increased doses of Sym004 induced The antiproliferative effects observed in vitro were also observed in potent antiproliferative effects in CtxR cell lines (Figure 2B), which de novo models of acquired resistance to cetuximab in vitro. De novo corresponded to more dramatic losses in total EGFR levels (Figure 3A); acquired resistant models were chosen for study because they more this finding suggests that loss of EGFR is a central mechanism by accurately portray the cellular events that result in cetuximab resistance which proliferation is inhibited. Treatment of CtxR cells with cetuximab in the clinic. In this model, CtxR xenograft tumors were significantly had no effect on EGFR levels and slightly enhanced EGFR activity growth delayed upon treatment with Sym004 (Figure 6A)comparedto (Figure 3B). Mandic et al. reported a similar phenomenon in a variety CtxR tumors that were continued on cetuximab therapy. The fact that of different HNSCC cell lines treated with cetuximab [27], indicating 67% of CtxR tumors put on Sym004 yielded tumor growth delay that cetuximab may function as a weak ligand in some cases. While demonstrates a unique property of Sym004 inhibition rather than just the current CtxR model has increased dependency on the EGFR, other spontaneous reduction in tumor size. While tumor shrinkage was models of CtxR have undergone oncogenic shift to other receptor tyro- observed in some mice upon Sym004 treatment, most experienced sine kinases (RTKs) [28] or constitutive activation of EGFR down- a delay in growth; growth delay in tumors of this size (>1000 mm3) stream effector molecules [29]; thus, Sym004 may not be an effective demonstrates that Sym004 can robustly inhibit proliferation, which treatment option for all CtxR tumors. Overall, EGFR-dependent CtxR may translate into potent antitumor effects in human patients. Addi- clones can be effectively targeted with Sym004 through the robust tionally, Sym004 treatment led to robust losses of EGFR and Ki67 degradation of EGFR and loss of downstream signaling emanating from staining, while there were minimal changes in cleaved caspase-3 levels this receptor. (Figure 6C ). Collectively, the antitumor effects of Sym004 observed Previous studies in our laboratory have indicated that the CtxR in this model of CtxR may be exerted through modulations in pro- clones used in this study have increased expression of EGFR ligands liferation pathways rather than apoptosis pathways. [8]. Further experimentation demonstrated that addition of EGFR Determining why patients with cancer become resistant to anti- ligands to the heparin binding (HP) CtxS cell line could enhance resis- EGFR therapeutics is a major clinical challenge. Data from the pres- tance to cetuximab. This phenomenon was observed by Hatakeyama ent study suggest that CtxR tumors may still be addicted to EGFR et al., where increased expression of HB-EGF was detected in intrinsic signaling, and therefore, using Sym004 may be of great clinical CtxR HNSCC cell lines and in tumor samples from patients with re- promise. Degradation of the EGFR, rather than inactivation, is a current disease [30]. Pedersen et al. supported these findings by report- powerful anticancer strategy because both kinase-dependent and Neoplasia Vol. 15, No. 10, 2013 Sym004 Can Overcome Resistance to Cetuximab Iida et al. 1205 independent functions of EGFR will be abolished. This therapeutic receptor antibody mixture with superior anticancer efficacy. Cancer Res 70, – may also have antitumor effects in the setting of EGFR-activating 588 597. 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